Periodic Reporting for period 1 - WallSATisFaction (The impact of SA signaling in CWI impairment and downstream factors)
Okres sprawozdawczy: 2023-04-01 do 2025-09-30
Plant survival and productivity depend on their ability to sense and adapt to constantly changing environmental conditions. Drought, osmotic stress, and mechanical strain threaten plant growth daily. They use a complex signaling networks to monitor the challenges and initiate adaptive responses to ensure their survival. Among the networks, cell-wall integrity (CWI) maintenance mechanism is fundamentally important because it ensure functional integrity of the cell wall during exposure to stress. If the cell wall is weakened or stressed, receptor-like kinases (RLKs) detect the weakness and trigger adaptive modification sin cellular and cell wall metabolism to restore integrity and strengthen resilience.
The WallSATisFaction project addressed a key knowledge gap by studying how the receptor-like kinase THESEUS1 (THE1) mediates CWI signaling and how this pathway regulates salicylic acid (SA) induction, transcriptional reprogramming, and primary cell-wall metabolism.
The project pursued three main scientific objectives:
- Identify signal transduction elements involved in SA induction upon CWI impairment.
- Discover novel transcriptional regulators of primary wall metabolism and stress adaptation.
- Characterize the molecular mode of action of these regulators and their target networks.
This research directly supports European and global sustainability goals. CWI signalling enables plants to adapt to environmental stress, which influences crop performance. This determines in turn agricultural productivity, resource efficiency, and ecological stability. The project therefore contributes to the European Green Deal, the Farm-to-Fork Strategy, and the EU Biodiversity Strategy for 2030, all of which prioritize sustainable agriculture, food security, and resilience to climate change.
The WallSATisFaction project addressed a key knowledge gap by studying how the receptor-like kinase THESEUS1 (THE1) mediates CWI signaling and how this pathway regulates salicylic acid (SA) induction, transcriptional reprogramming, and primary cell-wall metabolism.
The project pursued three main scientific objectives:
- Identify signal transduction elements involved in SA induction upon CWI impairment.
- Discover novel transcriptional regulators of primary wall metabolism and stress adaptation.
- Characterize the molecular mode of action of these regulators and their target networks.
This research directly supports European and global sustainability goals. CWI signalling enables plants to adapt to environmental stress, which influences crop performance. This determines in turn agricultural productivity, resource efficiency, and ecological stability. The project therefore contributes to the European Green Deal, the Farm-to-Fork Strategy, and the EU Biodiversity Strategy for 2030, all of which prioritize sustainable agriculture, food security, and resilience to climate change.
The project was structured around two research Work Packages (WPs), combining advanced molecular biology, omics technologies, and functional genetics in Arabidopsis thaliana.
WP1 – Selection of candidates focused on identifying genes regulated by THE1 during stress. Transcriptomic and phosphoproteomic datasets generated under cell-wall and osmotic stress were analyzed to identify candidate genes acting downstream of THE1. RNA-seq comparisons between the1 mutant alleles and wild-type plants treated with the cellulose biosynthesis inhibitor isoxaben or subjected to hyperosmotic stress identified 15 transcription factors (TFs) whose expression or phosphorylation was THE1-dependent.
The phosphoproteomics approach, initially planned as a complementary method, was expanded to provide post-translational insights beyond the original Description of Action (DoA). This effort generated a valuable dataset revealing phosphorylation-based regulation of signaling elements, substantially strengthening the project’s mechanistic framework.
WP2 – Functional characterization of candidates validated these findings experimentally. Fifteen T-DNA insertional mutant lines (12 knockouts, 2 knockdowns, and 1 over-expression line) were obtained and verified.
The following functional assays were performed to understand the functions of the TFs in plant growth, stress responses and cell wall metabolism:
Root growth phenotyping in medium containing isoxaben or sorbitol, identified altered sensitivity in multiple mutants; Cell-wall monosaccharide profiling revealed changes in glucose, rhamnose, galacturonic acid, and fucose content in particular TF mutants; qRT-PCR based expression analysis of genes required for cellulose biosynthesis (CesA1, CesA3, CesA6) and candidate TFs showed how certain TFs are required for moulating CESA gene expression; Phytohormone quantification (SA, JA, ABA) and lignin deposition assays identified TFs required for deposition of lignin and production of JA, thus connecting TFs required for transcriptional regulation with stress response and cell wall metabolism.
These studies demonstrated that several transcription factors act as stress-specific regulators of cell-wall metabolism and growth adaptation. Constructs for four key TFs were successfully generated to allow mechanistic experiments such as co-immunoprecipitation (co-IP) and chromatin-immunoprecipitation (ChIP). Unfortunately they could not be used for analysis in planta within the fellowship period due to the generation time of Arabidopsis.
Together, the integration of transcriptomics, phosphoproteomics, and mutant phenotyping provided a comprehensive understanding of THE1-dependent transcriptional regulation and established a strong foundation for future mechanistic and translational studies.
WP1 – Selection of candidates focused on identifying genes regulated by THE1 during stress. Transcriptomic and phosphoproteomic datasets generated under cell-wall and osmotic stress were analyzed to identify candidate genes acting downstream of THE1. RNA-seq comparisons between the1 mutant alleles and wild-type plants treated with the cellulose biosynthesis inhibitor isoxaben or subjected to hyperosmotic stress identified 15 transcription factors (TFs) whose expression or phosphorylation was THE1-dependent.
The phosphoproteomics approach, initially planned as a complementary method, was expanded to provide post-translational insights beyond the original Description of Action (DoA). This effort generated a valuable dataset revealing phosphorylation-based regulation of signaling elements, substantially strengthening the project’s mechanistic framework.
WP2 – Functional characterization of candidates validated these findings experimentally. Fifteen T-DNA insertional mutant lines (12 knockouts, 2 knockdowns, and 1 over-expression line) were obtained and verified.
The following functional assays were performed to understand the functions of the TFs in plant growth, stress responses and cell wall metabolism:
Root growth phenotyping in medium containing isoxaben or sorbitol, identified altered sensitivity in multiple mutants; Cell-wall monosaccharide profiling revealed changes in glucose, rhamnose, galacturonic acid, and fucose content in particular TF mutants; qRT-PCR based expression analysis of genes required for cellulose biosynthesis (CesA1, CesA3, CesA6) and candidate TFs showed how certain TFs are required for moulating CESA gene expression; Phytohormone quantification (SA, JA, ABA) and lignin deposition assays identified TFs required for deposition of lignin and production of JA, thus connecting TFs required for transcriptional regulation with stress response and cell wall metabolism.
These studies demonstrated that several transcription factors act as stress-specific regulators of cell-wall metabolism and growth adaptation. Constructs for four key TFs were successfully generated to allow mechanistic experiments such as co-immunoprecipitation (co-IP) and chromatin-immunoprecipitation (ChIP). Unfortunately they could not be used for analysis in planta within the fellowship period due to the generation time of Arabidopsis.
Together, the integration of transcriptomics, phosphoproteomics, and mutant phenotyping provided a comprehensive understanding of THE1-dependent transcriptional regulation and established a strong foundation for future mechanistic and translational studies.
The WallSATisFaction project advanced the state of the art in plant cell-wall biology and stress signalling. Prior to this work, the role of THE1 was primarily defined at the phenotypic and early stimulus perception/signaling level, with very limited knowledge of the downstream molecular network responsible for translating the early signaling in adaptive responses. This project started to close the previously existing knowledge gap by combining transcriptomics data with functional analyses of candidate genes to characterize the transcriptional network controlling the molecular mechanisms responsible for plant adaption to environmental stress causing cell wall damage.
Key discoveries include:
- Identification and characterization of novel transcription factors, acting downstream of THE1, regulating cell-wall metabolism and hormone-mediated stress adaptation.
- Generation of a unique phosphoproteomics dataset identifying phosphorylation events caused by cell wall damage and osmotic stress, providing unprecedented insight into post-translational regulation in CWI signaling.
- Creation of a validated mutant collection and molecular constructs that enable future in-depth mechanistic studies within the host group’s ERC Synergy project Hydrosensing.
These findings provide a mechanistic framework for understanding how plants integrate signals deriving from mechanical and hyper-osmotic stress into adaptive growth and defense responses. The outcomes also have translational potential, because they can inform future strategies aimed at developing food crop varieties with enhanced resistance to environmental stress and resource-use efficiency.
All project datasets have been deposited in public repositories and will be shared under FAIR data principles upon publication of the manuscripts currently in preparation, ensuring transparency and long-term accessibility to the scientific community while also maximizing the probability of generating useful outputs.
Key discoveries include:
- Identification and characterization of novel transcription factors, acting downstream of THE1, regulating cell-wall metabolism and hormone-mediated stress adaptation.
- Generation of a unique phosphoproteomics dataset identifying phosphorylation events caused by cell wall damage and osmotic stress, providing unprecedented insight into post-translational regulation in CWI signaling.
- Creation of a validated mutant collection and molecular constructs that enable future in-depth mechanistic studies within the host group’s ERC Synergy project Hydrosensing.
These findings provide a mechanistic framework for understanding how plants integrate signals deriving from mechanical and hyper-osmotic stress into adaptive growth and defense responses. The outcomes also have translational potential, because they can inform future strategies aimed at developing food crop varieties with enhanced resistance to environmental stress and resource-use efficiency.
All project datasets have been deposited in public repositories and will be shared under FAIR data principles upon publication of the manuscripts currently in preparation, ensuring transparency and long-term accessibility to the scientific community while also maximizing the probability of generating useful outputs.